Microbial process for preparing pravastatin

ABSTRACT

The present invention relates to a new microbial process for the preparation of the compound formula (I)                    
     from a compound of general formula (II)                    
     wherein R stands for an alkali metal or ammonium ion, by the submerged cultivation of a mold strain able to 6β-hydroxylate a compound of the Formula (II) in aerobic fermentation and by the separation and purification of the product of Formula (I) formed in the course of the bioconversion. The process comprises cultivating a strain of  Mortierella maculata  filamentous mold species that is able to 6β-hydroxylate a compound of the general Formula (II), on a nutrient medium containing assimilable carbon and nitrogen sources and mineral salts and separating the product formed from the fermentation broth, then isolating the compound of formula (I) and purifying the same. Novel strains of  Mortierella maculata  are also disclosed.

This application claims benefit of Provisional No. 60/118,458 filed Feb.3, 1999 and Provisional No. 60/134,759 filed May 18, 1999.

FIELD OF THE INVENTION

The present invention relates to a process for the preparation ofpravastatin, and particularly to a microbial process for the manufactureof pravastatin on an industrial scale.

BACKGROUND OF THE INVENTION

The highest risk factor of atherosclerosis and especially coronaryocclusion is the high cholesterol level of the plasma. In the last twodecades 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC.1.1.1.34) asthe rate limiting key enzyme of the cholesterol biosynthesis wasextensively examined. Pravastatin, a compound of Formula I,

and other related compounds (compactin, mevinolin, simvastatin) are thecompetitive inhibitors of the HMG-CoA reductase enzyme [A. Endo et al.,J. Antibiot. 29, 1346-1348 (1976); A. Endo et al., FEBS Lett. 72,323-326 (1976); C. H. Kuo et al., J. Org. Chem. 48, 1991 (1983)].

Pravastatin was first isolated by M. Tanaka et al. (unpublished results)from the urine of a dog during the examination of the compactinmetabolism (Arai, M. et al., Sankyo Kenkyusyo Nenpo, 40, 1-38, 1988).Currently pravastatin is a cholesterol lowering agent with the mostadvantageous action mechanism in the therapy. Its most importantcharacter is tissue selectivity, i.e., it inhibits the cholesterolsynthesis at the two main sites of the cholesterogenesis, such as in theliver and in the small intestine, while in other organs theintracellular enzyme limiting effect is hardly detectable, At the sametime the cholesterol biosynthesis limiting effect of mevinolin andsimvastatin is significant in most of the organs (T. Koga et al.,Biochim. Biophys. Acta, 1045, 115-120, 1990).

Pravastatin essentially differs in chemical structure from mevinolin andsimvastatin which have more lipophilic character. In the case of thelatter compounds the substituent connected to the C-1 carbon atom of thehexahydronaphthalene skeleton is ended in a 6-membered lactone ring,while in the case of pravastatin, instead of the lactone ring, thebiologically active, opened dihydroxy acid sodium salt form is present.Another important structural difference is that instead of the methylgroup of mevinolin and simvastatin at the C-6-position of thehexahydronaphthalene ring, a hydroxyl group can be found in pravastatin,which results in a further increase in its hydrophilic character.

As a result of the above structural differences pravastatin is able topenetrate through the lipophilic membrane of the peripheral cells onlyto a minimal extent (A. T. M., Serajuddin et al., J. Pharm. Sci. 80,830-834, 1991).

Industrial production of pravastatin can be achieved by two fermentationprocesses. In the first, microbiological stage compactin is prepared,then in the course of a second fermentation the sodium salt of compactinacid as a substrate is converted to pravastatin by microbialhydroxylation at the 6β-position.

According to published patents, the microbial hydroxylation of compactincan be accomplished to various extents with mold species belonging todifferent genera, and with filamentous bacteria belonging to theNocardia genus, with Actinomadura and Streptomyces genera (Belgianpatent specification No. 895090, Japanese patent specification No.5,810,572, U.S. Pat. Nos. 4,537,859 and 4,346,227 and published Europeanpatent application No. 0605230). The bioconversion of compactinsubstrate was published in a 500 μg/ml concentration using filamentousmolds such as Mucor hiemalis, Syncephalastrum nigricans, Cunninghamellaechinulata and in 2000-4000 μg/ml with Nocardia, Actinomodura andStreptomyces strains belonging to the prokaryotes.

A general problem experienced in the cases of manufacturing thepravastatin with filamentous molds is that due to the antifungal effectof compactin, the microorganisms are not able to tolerate the compactinsubstrate fed to the culture even at low concentrations (Serizawa etal., J. Antibiotics, 36, 887-891, 1983). The cell toxicity of thissubstrate was also observed in the hydroxylation with Streptomycescarbophilus extensively studied by Japanese researchers (M. Hosobuchi etal., Biotechnology and Bioengineering, 42, 815-820, 1993).

Japanese authors tried to improve the hydroxylating ability of theStreptomyces carbophilus strain with recombinant DNA techniques. Acytochrome P-450 monooxygenase system is needed for the hydroxylation ofcompactin (Matsuoka et al., Eur. J. Biochem. 184, 707-713, 1989).However, according to the authors, in the bacterial cytochrome P450monooxygenase system not one but several proteins act in the electrontransport, which aggravate the application of the DNA techniques.Development of a cost-effective microbiological hydroxylation method forthe manufacture of pravastatin is an extremely difficult, complex task.

The aim of the present invention is to elaborate a new microbial processfor the preparation of pravastatin from compactin in industrial scale,which would produce pravastatin at more advantageous conditions thanthose previously known. During our research work, above all we tried tofind a microorganism strain with a hydroxylase enzyme that can beadapted for the microbial transformation of compactin to pravastatin ina high concentration.

SUMMARY OF THE INVENTION

The present invention relates to a microbial process for the preparationof the compound of formula (I)

from a substrate compound of formula (II),

wherein R stands for an alkali metal or ammonium ion, comprising thesteps of (a) cultivating a strain of Mortierella maculata filamentousmold species able to 6β-hydroxylate a compound of formula (II) on anutrient medium containing assimilable carbon- and nitrogen sources andmineral salts, (b) feeding the substrate to be transformed into thedeveloped culture of Mortierella maculata, (c) fermenting the substrateuntil the end of bioconversion, (d) separating the compound of formula(I) from the culture broth, and (e) isolating the compound of formula(I).

The present invention also relates to a biologically pure culture of theMortierella maculata n. sp. E97 strain deposited at the NationalCollection of Agricultural and Industrial Microorganisms, Budapest,Hungary on Jul. 24, 1998 under the number NCAIM(P)F 001266 abiologically pure culture of its mutant, the Mortierella maculata n. sp.E97/15/13 strain deposited at the National Collection of Agriculturaland Industrial Microorganisms, Budapest, Hungary on Jul. 24, 1998 underthe number NCAIM(P)F 001267.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is an illustration of the physical characteristics of Mortierellamaculata n. sp. E-97.

DETAILED DESCRIPTION OF THE INVENTION

In the course of our screening program, which covered about 5500prokaryotic and eukaryotic strains, 23 microorganisms were selected,which were able to hydroxylate compactin in opposition. Among thesestrains a filamentous mold proved to be more appropriate for theproduction of pravastatin due to its higher resistance against compactinas compared to the strains known from published patents. According tothe taxonomic investigation, this strain proved to be a newrepresentative of the species belonging to the Mortierella genus(Mortierella maculata n. sp.). From the selected molds a new strain wasisolated on the one hand by the application of the mutation-selectionmethods, and on the other hand by the induction of the hydroxylaseenzyme of the strain, which one was able to hydroxylate the compactinsubstrate to pravastatin in a higher concentration than published sofar. As mutagenic agents, physical and chemical mutagens were applied(UV irradiation, methyl methane sulfonate,N-methyl-N′-nitro-N-nitrosoguanidine). After the mutagenic treatments,in order to prepare haploid cells, the spore suspension was spread onbenomyl-containing agar plates, then in order to induce the hydroxylaseenzyme the developed colonies were inoculated onto 100 μg/ml8-de-(2-methyl-butyryl)-compactin-containing or compactin-containingagar plates. By the application of these methods a mutant strain wasprepared from the new strain that is able to convert compactin topravastatin to a significantly higher extent than the parent strain.

In the course of the optimizing experiments we determined thecomposition of the most beneficial inoculum, and the most advantageousbioconversion media for the compactin hydroxylation, as well as theoptimal method for the repeated feeding of compactin in a highconcentration.

Consequently, this invention is based on the recognition that the E-97and E-97/15/13 designated strains of the isolated mold named Mortierellamaculata, which were deposited under accession numbers of NCAIM(P)F001266 and NCAIM(P)F 001267 respectively, at the National Collection ofAgricultural and Industrial Microorganisms (Department of Microbiologyand Biotechnology, University of Horticulture and the Food IndustryBudapest), under appropriate fermentation conditions are able tomanufacture pravastatin to a high extent, while the undesired relatedcompounds such as the acid forms of 6α-hydroxy-compactin,2α-hydroxy-compactin, 8-de-(2-methyl-butyryl)-compactin,3α,5β-dihydroxy-5,6-dihydro-isocompactin, 8a,β-hydroxy-compactin and thehydroxylated derivatives at positions 2 and 3 of the 2-methyl-butyrylside chain of compactin are obtained only in small or trace amountsduring the bioconversion. Thus, these strains are especially appropriatefor manufacturing pravastatin in an industrial scale.

Taking into account that the economical manufacture of the activeingredient on an industrial scale is a function of the compactinsubstrate concentration, it is important to have a strain that is ableto tolerate high compactin and pravastatin concentrations. Consequently,a further important part of the invention is the recognition that thehydroxylating ability of the original mold isolate can be improved bythe application of mutation-selection and enzyme induction methods and,furthermore, that by the development of an appropriate method forsubstrate feeding the hydroxylation of large quantities of compactin topravastatin can be executed in a single procedure. In conclusion, thenew mutant strain designated as Mortierella maculata n. sp. E-97/15/13is especially appropriate for the manufacture of pravastatin.

Taxonomic features of the isolated new mold species comparing it to themost important diagnostic attributes of the known Mortierella speciesare summarized below.

Taxonomic Description of the Holotype Strain Mortierella maculata Nov.Spec. E-97

On starch-casein-malt extract-agar media the aerial mycelium is welldeveloped (more than 10 μm thick covering layer over the substratemycelium). At the beginning it appears as a tightly woven white web ofhyphae, in which later yellowish sporulating spots with a few mmdiameter sparsely appear (new name “maculatus” refers to the above:spotted). This yellowish coloration can sometimes occupy the largercontinuous surfaces of the aerial web. The color of the substratemycelium is on Czapek-, bloody-Czapek, tyrosine-, starch-casein-, maltextract-, etc. agar media mostly colorless or light yellowish. The colorof the substrate mycelial web is light reddish on yeastextract-glucose-peptone medium. Production of diffusible and solublepigments on the above listed media is not experienced, or only rarely aninsignificant yellowish coloration occurs on these media. Colonies ofstrain E97, due to their volatile oil production and similarly to manyother species of Mortierella, (except to species of section Isabellina),can exude a very characteristic strong scent.

Sporangiophores, designated by reference numerals 1-7 in FIG. 1,frequently develop locally on the aerial hyphae (but less on thesubstrate ones) in great numbers at very different distances from eachother. They are not branching, but are mostly straight or curved. Theirlength is generally between 60-80 μm. The starting point in theoverwhelming majority of cases is a more or less short but stronglyswollen hyphal section of the aerial web, from which they are separatedby walls. Sporangiophores themselves can be also swollen (sometimesstrongly), as shown by reference numeral 6, but in the direction of thesporangium they gradually narrow, from 5.0-9.0 μm to 1.0-2.0 μm. It isan important taxonomic character that below the sporangiophores theynever broaden out (see reference numeral 8).

Sporangia are spherical; in some cases slightly flattened spheres. Theirdiameter is about 6.0-17.0 μm, relatively small compared to the measuresof sporangia of other Mortierella species. Sporangia may contain manyspores, but sporangia bearing only one spore also exist. The spores 9are cylindrical or less oval. Their size is 3.0-5.0×1.5-2.0 μm. Withinthe individual spores one or two small dark spherical oil-drops 10 maybe present. Due to the very easy disintegration of the wall ofsporangia, in wet surroundings the spores will quickly be scattered.After the disintegration of the sporangium, sometimes at the end of thesporangiospores, a fine pitchfork-like “collar” and a very shortrudimental (and not typical) columella can be observed. Gemmae 15-28that are spherical or cylindrical may occur on most different diagnosticmedia. Usually the size is 10-25 μm. In the cultures chains of sphericalgemmae 13, budding cells, intercalated gemmae 15-23, hyphal associationsof particular spiral-growth of one hypha around the other 11,anastomotic-like structures and giant cells, etc. can also be found. Inthe aerial mycelium also large (50-250 pm diameter) very dense hyphalwebs 14 can be seen but without the presence of detectable zygotes.

Cultures of strain E-97 are able to reduce nitrates to nitrites, do nothydrolyze starch, esculin, arginine or gelatine but hydrolyze Tweenpolysorbates and do not decompose paraffin hydrocarbons. The cultures ofstrain E-97 have urease activity show a good growth between pH 7.0 and9.0 tolerate a maximum 2% NaCl. The effect of xanthine, hypoxanthine,lecithin, tyrosine and adenine are negative. A strong acid production ofthe cultures has been detected from glucose, fructose, glycerine andgalactose, but very weak or no production from xylose, arabinose,raffinose, sorbitol, inositol, inulin, etc. Weak growth is detected onpyruvate and acetate but no growth could be found with benzoate,salicylate, citrate, lactate, succinate, tartarate and malonate. A goodgrowth was observed with glucose and fructose as sole carbon-sources inthe medium. Utilization tests with xylose, arabinose, rhamnose, sucrose,raffinose, mannitol and inositol proved to be negative. The cultures donot decompose cellulose.

Systematic position: Strain E97 belongs to the family Mortierellaceaeand it is a typical member of the genus Mortierella: sporangia containgenerally many spores, columella is extremely reduced, gemmae arefrequently present, the occurrence of zygotes has not been detected andthe colonies exude a very characteristic strong scent. Within the genusMortierella, strain E-97 is a typical representative of the “SectionAlpina.” The latter can be characterized by very short non-branchingsporangiophores (maximal length to 200 μm), and minute sporangia (Zycha,H. und Siepmann, R, Mucorales. Eme Beschreibung aller Gattungen undArten dieser Pilzgruppe. D-3301 Lehre, Verl. von J. Cramer. 1969). Amongthe members of the section Alpina, strain E-97 shows the greatestsimilarity to the species M. thaxteri Björling 1936 and M. renisporaDixon-Stewart 1932. However, the data in the Table I clearly show thedifferences in diagnostic properties of strain E-97 and of these twospecies. Accordingly, as a new species herewith we introduce the strainunder the name Mortierella maculata nov. spec. E-97.

TABLE 1 A comparison of the holotype strain E-97 of Mortierella maculatan. sp. with the species M. renispora and M. thaxteri on the basis of keydiagnostic properties Mortierella renispora Mortierella strain E-97Mortierella thaxteri Origin of Ordinary, however the Laterally from(swollen or Laterally from swollen separated sporangiophores hyphae arewider than the normal) aerial hyphae or not segments of aerial hyphae ornot regular ones, laterally from separated sections of substrateseparated segments of substrate hyphae broadened swollen hyphae. hyphae.Shape and size of Gradually decreasing towards Mostly straight orcurved, not Length about 60-90 μm. Width at the sporangiophores the topfrom 10 μm to 3 μm. branching. Width gradually starting point is about5-7 μm, at the tip Length is about 200 μm. decreasing towards the tip:it is reduced to 1.5-2 μm. Immediately from 5-9 μm to 1.5-2.5 μm. underthe sporangium broadened out Length is about 60-80 μm. At the tip neverbroadened. Shape and size of Colorless, diameter is 25 μm. Mostlyspherical (6-17 μm Spherical (12-20 μm diam.). They sporangia diam.) butrarely less flat- contain many spores but on certain tened. Generallycontain many media there is also only one spore- spores, rarely onespore. bearing sporangra. Wall (membrane) Spreading membrane. AfterDisintegrating. A pitchfork- Disintegrating, a minute backward- of thesporangia disintegration remains a like collar remains. bending collarremains. collar-like structure. Shape and size of Roughly kidney-shaped.Cylindrical. Length (3-5 μm) Ellipsoidal hyaline spores of 3.5-4 × thespores hyaline structures, sizes are can doubly exceed the width 1.5-2μm) dimension. 2 × 4 μm. (1.5-2 μm). Gemmae Occur on the most differentFrequent on very different Intercalary, oval gemmae (10-14 μm) in mediamedia, mostly in the aerial the substrate mycelium. mycelium. Sphericalor elongated (10-25μm). Zygotes Frequent on all diagnostic Not detected.Not observed. media. Diameter together with the covering hyphae is about500 μm, without them about 30 μm. Dense foci of the Large densely wovenhyphal In old cultures large (100-125 μm hyphal web foci (50-250 μm)frequent but diam) yellowish-grey dense hyphal without zygotes. webs,without zygote. Color and habit of Loose, always white hyphal White withyellowish spots At first spider's web-like, later more the aerialmycelium web dense.

In the process for the preparation of pravastatin according to thepresent invention, preferably the culture of the mold strain designatedas Mortierella maculata n. sp. E97 or its mutant designated as E97/15/13is used. The selected strain is highly advantageous due to its fastgrowth. As a carbon source it easily utilizes glucose, glycerine,fructose, or galactose. As a nitrogen source yeast extract, peptone,casein, meat extract, soybean meal, corn steep liquor, sodium nitrate,or ammonium sulfate can be used.

In the culture media used for the production of pravastatin besides theabove carbon and nitrogen sources mineral salts, e.g., potassiumdihydrogen phosphate, magnesium chloride, magnesium sulfate, traceelements (ferrous, manganous salts), amino acids and antifoaming agents,can be present.

According to a preferred embodiment of the present invention, the sporesuspension having been prepared from the slant agar culture of theMortierella maculata n. sp. designated as E-97 strain or its mutant[NCAIM(P)F 001267] designated as E97/15/13, is seeded into an inoculummedium; then 10% of the inoculum culture, which is cultivated for 3 daysat about 25-30° C., preferably at about 24-28° C., most preferably atabout 28° C., is transferred into the bioconversion medium. Then it isincubated for 4 days at about 25-28° C., preferably at about 28° C.,then glucose and the sodium salt of compactin acid are fed into theculture. Depending on the concentration of the fed compactin substrate,the cultivation is continued for 2-12 days further under aerobicconditions, while the pH is maintained between 5.5 and 7.5, preferablyat 7.0. The bioconversion is done under stirred and aerated conditions,when the air flow rate is 0.2 vvm, the spinning rate of the stirrer is400/min.

In the course of the fermentation the bioconversion of compactinsubstrate was followed by a high pressure liquid chromatographic method(HPLC). According to this method, the sample of the broth is dilutedtwofold with methanol and centrifuged, and the supernatant is used forthe HPLC analysis under the following parameters: Waters analytical HPLCequipment; column: Nucleosil C₁₈ 10 μm; detection wavelength: 238 nm;injection volume: 20 μl, flow rate: 1 ml/min; gradient elution is used,eluents: A=0.05% aqueous solution of phosphoric acid, B=acetonitrile.

Elution Gradient:

Time (min) Eluent A (%) Eluent B (%) 0 70 30 20 0 100 25 0 100 25.1 7030 35 70 30

Approximate retention times: pravastatin 8.6-9.0 mm; compactin acid11.6-12.0 mm; pravastatin lactone 15.0-15.5 mm, compactin 16.5-17.0 mm.

For the production of pravastatin the aqueous solution of the sodiumsalt of compactin acid is added at the 96th hour of the cultivation. Forthis procedure the substrate is prepared in solid form as follows.Compactin lactone is hydrolyzed in a 0.2M sodium hydroxide solution for2 hours at 40° C., then the pH of the reaction mixture is adjusted to7.5 by hydrochloric acid and the neutralized solution is layered on aDiaion HP-20 adsorbent column; the sodium chloride formed during theneutralization is eliminated by aqueous washing of the column, and thenthe sodium salt of the compactin acid is eluted from the column by 50%aqueous acetone. Thereafter the eluate is distilled in vacuum and theaqueous residue is lyophilized. After neutralization the aqueoussolution of the alkaline hydrolysate of compactin can also be directlyused as substrate. In this case the compactin acid sodium salt contentof the hydrolysate is measured by HPLC, and the solution is kept at −20°C. until being applied.

The higher the broth pH reached by the fourth day of the fermentation,the more advantageous for the hydroxylation of the compactin substrate.Feeding of the compactin substrate is allowed to be started when the pHof the broth exceeds 6.3. At the 4th day of the fermentation as much ofthe sterile filtered aqueous solution of compactin acid sodium salt isadded as needed to reach the 500 μg/ml concentration. Glucose is alsofed to the culture from its 50% solution sterilized at 121° C. for 25minutes as follows: if the pH of the broth is higher than 6.7 value, 1%glucose is added related to the volume of the broth, while if the pH iswithin the 6.3-6.7 range the quantity of the glucose fed is 0.5%.Compactin acid sodium salt is consumed from the broth after 24 hours,its transformation is analyzed by HPLC measurement. In this case, foreach ml of the broth another 500 μg of compactin is added. Besides thecompactin substrate, glucose is also fed as described above. Morphologyof the 120 hour culture is characterized by the small pellet growth(diameter of the pellet; 0.5-3.0 mm). After 24 hours the second dose ofsubstrate is also consumed from the broth, thus a further portion ofcompactin acid sodium salt producing a 500 μg/ml concentration of it inthe whole broth is added parallel with the glucose feeding dependent onthe pH value of the broth. From the 4th day of the fermentation thesubstrate and the glucose feeding is repeated in daily frequency as itis written before until the 17th-18th day of the fermentation.

For the recovery of the product from the broth, it is advantageous totake into consideration the fact that during the bioconversionpravastatin is formed in its acidic form, thus it can be isolated fromthe filtrate of the broth by its adsorption on an anion exchange resincolumn. For the isolation of the product it is advantageous to use astrongly basic anion exchange resin which is apolystyrene-divinylbenzene polymer carrying quaternary ammonium activegroups. The product can be adsorbed directly from the filtrate of thebroth by mixing the anion exchange resin being in hydroxyl form into it.The product being adsorbed on the ion exchange resin can be eluted fromthe column by acetic acid or a sodium chloride-containing acetone-watermixture, preferably 1% sodium chloride containing acetone-water (1:1)mixture. Pravastatin-containing fractions are combined and the acetonebeing in the eluate is distilled off in vacuum. The pH of theconcentrate is adjusted with 15% sulfuric acid into the range of 3.5-4.0and the aqueous solution is extracted by ethyl acetate. The ethylacetate extract is washed with water and dried with anhydrous sodiumsulphate. Then the lactone derivative is prepared from pravastatin. Thelactone ring closure is carried out in dried ethyl acetate solution atroom temperature, under continuous stirring by inducing the lactoneformation with trifluoroacetic acid being present in catalytic quantity.The transformation procedure is checked by thin layer chromatographicanalysis (TLC). After finishing the lactone formation the ethyl acetatesolution is washed at first with 5% aqueous sodium hydrogen carbonatesolution and then with water, then it is dried with anhydrous sodiumsulfate and evaporated in vacuum. The evaporated residue is treated inacetone solution with charcoal, then evaporated again and recrystallizedfrom a 1-4 carbon atom-containing aliphatic alcohol, preferably fromethanol. The evaporation residue of the recrystallization mother liquoris purified with silica gel column chromatography applying the mixtureof ethyl acetate-n-hexane with gradually increasing ethyl acetatecontent as the eluent.

From the pravastatin lactone obtained after recrystallization andchromatographic purification pravastatin is prepared by hydrolysis atroom temperature in acetone with equivalent quantity of sodiumhydroxide. When the pravastatin sodium salt formation is completed, thereaction mixture is diluted with water and neutralized, and the acetonecontent is distilled in vacuum. Pravastatin is adsorbed from theobtained aqueous residue on a Diaion HP-20 resin-containing column,washed with deionized water and eluted from the column with anacetone-deionized water mixture. Then the pravastatin containingfractions are combined, the acetone content is distilled off and afterthe lyophilization of aqueous residue pravastatin can be obtained inhigh purity, which can be recrystallized from an ethyl acetate-ethanolmixture.

In the course of the procedure the whole quantity of pravastatin can beadsorbed During the lactone closure of pravastatin3α-hydroxy-iso-compactin and other by-products can also be formed.Although these latter reactions decrease the yield of the isolation butthose compounds can be separated by the above-described purificationmethod and consequently, pravastatin can be manufactured this way in apharmaceutically acceptable quality.

After finishing the bioconversion pravastatin can be extracted eitherfrom the fermentation broth or from the filtrate obtained after theseparation of the filamentous mold cells. Filamentous mold cells can beeliminated either by filtration or centrifugation; however, it isadvantageous especially on an industrial scale to make a whole brothextraction. Before extraction the pH of either the fermentation broth orthe filtrate of the broth is adjusted to 3.5-3.7 with a mineral acidpreferably with diluted sulfuric acid. The extraction is done with anester of acetic acid and a 24 carbon atom containing aliphatic alcohol,preferably with ethyl acetate or isobutyl acetate. Extraction stepsshould be done very quickly in order to avoid the formation of thelactone derivative from pravastatin at acidic pH.

From the organic solvent extract the pravastatin in acid form can betransferred as the sodium salt into the aqueous phase. For example, froman ethyl acetate extract pravastatin can be extracted by 1/10 and 1/20volume ratio of 5% sodium hydrogen carbonate or weakly alkaline water(pH 7.5-8.0). It was found that pravastatin can be recovered in a pureform from the above-obtained alkaline aqueous extract by columnchromatography with the application of a non-ionic adsorption resin. Anadvantageous method is to first remove the solvent dissolved in theaqueous phase by vacuum distillation from the alkaline aqueous extract,and then the aqueous extract is loaded on a Diaion HP-20 column.

Pravastatin sodium salt being adsorbed on the column is purified byelution increasing gradually the acetone content of the aqueoussolutions, then the pravastatin-containing main fractions are combinedand concentrated in vacuum. The aqueous concentrate is purified furtherby chromatography on another Diaion HP-20 column, obtaining an eluatecontaining pure pravastatin, from which after clarification withcharcoal and lyophilization pravastatin can be obtained in apharmaceutically acceptable quality.

This isolation procedure consists of fewer stages than the previous one,since the lactone formation of pravastatin and its hydrolysis are notinvolved in the procedure. During the isolation pravastatin is exposedto acidic condition for only a limited time, under which it is lessstable than in neutral or alkaline solutions, consequently, during thisisolation procedure artefacts are practically not formed.

Furthermore, it was found that the chromatography on Sephadex LH-20Dextran gel (hydroxypropylated derivative) is advantageously used forpurifying pravastatin. By application of this method pravastatinexceeding the purity of 99.5% (measured by HPLC) can be produced.

In the course of our experiments it has been recognized that from theorganic solvent extract, preferably from the ethyl acetate or isobutylacetate extract of the broth or the broth nitrate of the filamentousmold or the filamentous bacteria strains among them the Mortierellamaculata n. sp. strain able to 6β-hydroxylate a compound of generalformula (II), pravastatin can be precipitated as a crystalline salt withsecondary amines. Further it was found that for the salt formationseveral secondary amines containing alkyl, cycloalkyl-, aralkyl- oraryl-substituents are appropriate. Expediently non-toxic secondaryamines were selected among them, e.g., dioctylamine, dicyclohexylamine,dibenzylamine. The isolation of the organic secondary amine saltintermediates, e.g., the dibenzylamine salt was carried out by addingdibenzylamine in 1.5 equivalent quantity related to the pravastatincontent of the extract, then the extract is concentrated by vacuumdistillation to 5% of its original volume, then another quantity ofdibenzylamine is added into the concentrate in 0.2 equivalent ratio. Thecrystalline dibenzylamine salt is precipitated from the concentrate. Thecrystalline crude product is filtered and dried in vacuum. Then it isclarified with charcoal and recrystallized in acetone.

In the procedure mentioned earlier in which the organic solventextraction and the reextraction at alkaline pH are involved, theisolation method based on the secondary amine salt formation can be usedalso for the replacement of the column chromatographic purification. Inthis case it is advantageous to precipitate the pravastatindibenzylamine salt from the isobutyl acetate extract obtained after theacidification of the alkaline aqueous extract.

Pravastatin organic secondary amine salts can be transformed topravastatin by sodium hydroxide or a sodium alkoxide, preferably sodiumethoxide.

The transformation is detailed in the case of pravastatin dibenzylaminesalt. The recrystallized dibenzylamine salt is suspended in an isobutylacetate-water mixture, then equivalent quantity of sodium hydroxide isadded in aqueous solution to the suspension by maintaining understirring the pH in the range of 8.0-8.5. After disappearance of thesuspension the phases are separated and the pravastatin-containingaqueous solution is washed twice with isobutyl acetate. The aqueoussolution is clarified with activated carbon and lyophilized yieldingpravastatin in a pharmaceutically acceptable quality.

One preferred method for the transformation of pravastatin dibenzylaminesalt to pravastatin is to suspend the recrystallized dibenzylamine saltin ethanol, then equivalent quantity or small excess of sodium ethoxideis added under stirring to the suspension, then the reaction mixture isconcentrated in vacuum and by adding acetone the pravastatin isprecipitated in crystalline form from the concentrate.

Another preferred method for the transformation of pravastatindibenzylamine salt to pravastatin is to dissolve the recrystallizeddibenzylamine salt in ethyl acetate-ethanol mixture and by addingequivalent quantity or small excess of sodium hydroxide in ethanol tothe solution pravastatin is precipitated.

The isolation of pravastatin via a secondary amine salt intermediate isa simpler procedure than any previously known isolation procedures.During the procedure artifacts are not formed, and the separation ofpravastatin from the by-products of the bioconversion and from thevarious metabolic products biosynthesized by the hydroxylatingmicroorganism can be solved without the application of anychromatographic methods.

The structures of pravastatin, pravastatin lactone and the isolatedsecondary amine salts of pravastatin have been proven by UV, IR, ¹H-NMR,¹³C-NMR and mass spectroscopic methods.

EXAMPLES

The invention Will be more fully described and understood with referenceto the following examples, which are given by way of illustration andare not intended to limit the scope of the invention in any way.

Example 1

A spore suspension was prepared with 5 ml of a 0.9% sodium chloridesolution obtained from a 7-10 day old, malt extract-yeast extract agarslant culture of Mortierella maculata nov. spec. E-97 [NCAIM(P)F 001266]strain able to 6β-hydroxylate compactin and the suspension was used toinoculate 100 ml inoculum medium PI sterilized in a 500 ml Erlenmeyerflask.

Composition of the Medium PI:

glucose 50 g soybean meal 20 g in 1000 ml tap water.

Before the sterilization the pH of the medium was adjusted to 7.0, thenit was sterilized at 121° C. for 25 min. The culture was shaken on arotary shaker (250 rpm, 2.5 cm amplitude) for 3 days at 28° C., then 10ml of the obtained culture was transferred into 100-100 ml bioconversionmedia MU/4 sterilized in 500 ml Erlenmeyer flask for 25 min at 121° C.

Composition of the Medium MU/4:

glucose 40 g soybean meal 20 g casein-peptone  1 g asparagine  2 gpotassium dihydrogen phosphate 0.5 g  in 1000 ml tap-water.

Before the sterilization the pH of the medium was adjusted to 7.0, thenit was sterilized at 121° C. for 25 min.

Flasks were shaken on a rotary shaker (250 rpm, 2.5 cm amplitude) for 4.days at 25° C., then 50-50 mg compactin substrate (compactin acid sodiumsalt) was added in sterile-filtered aqueous form into the cultures, thenthe cultivation was continued. Similarly, at the 5th day another 50-50mg compactin acid sodium salt was added into the mold cultures, and thefermentation was continued for a further 24 hours. The pravastatincontent of the broth was determined by HPLC. The fermentation wascontinued for 168 hours. At the end of the bioconversion the averagepravastatin concentration of the fermentation broth was 620 μg/ml.

Example 2

In a laboratory scale fermenter with 5 liters working volume a MU/Sbioconversion culture medium is prepared, the components of the culturemedium are added corresponding to 5 liters, volume but it was loaded uponly to 4.5 liters, then it was sterilized for 45 min at 121° C. andseeded with 500 ml of the inoculum culture made according to the Example1.

Composition of Medium MU/8:

glucose 20 g glycerine 20 g soybean meal 20 g peptone  5 g potassiumdihydrogen phosphate 0.5 g  polypropyleneglycol 2000  1 g in 1000 ml tapwater.

Before sterilization the pH of the medium was adjusted to 7.0 value.

The fermentation was carried out at 28° C., with a stirring rate of 400rpm and with an aeration rate from bottom direction 60 liters/hour for 4days. At the 2nd day after the transfer the culture started to foamheavily, which can be decreased by the addition of furtherpolypropyleneglycol 2000. At the beginning of the fermentation (16-20hours) the pH decreased from the initial value of 6.5 to 5.0-5.5, thenfrom the 3rd day it started to increase and reached 6.3-7.5 by the 4thday. The feeding of the compactin substrate is allowed to be started ifthe pH of the broth is above 6.3. At the 4th day of the fermentation 2.5g compactin substrate is added in sterile filtered aqueous solution.Calculated for the volume of the broth 0.5-1.0% glucose was added intothe culture depending on the pH in the form of 50% solution sterilizedat 121° C. for 25 minutes in parallel with the substrate feeding. After24 hours the compactin substrate is consumed from the culture, which isdetected by HPLC from the samples taken from the fermenter. In this caseanother 2.5 g compactin substrate and glucose were added as describedabove, and the bioconversion was continued for 24 hours further when thesubstrate was converted to pravastatin.

After finishing the fermentation, 5.1 liters broth containing 630 μg/mlpravastatin were filtered on a filter cloth. Two liters water were addedto the separated mycelium, then the mycelium suspension was stirred forone hour and filtered. These two filtrates were combined and passedthrough with a flow rate of 500 ml/hour on a column containing 138 g(250 ml) Dowex Al 400 (OH) resin (diameter of the column 3.4 cm, heightof the resin bed: 28 cm), then the resin bed was washed with 300 mldeionized water. Subsequently, the elution from the resin was carriedout by 1 liter acetone-water (1:1) mixture containing 10 g sodiumchloride. The volume of the fractions was 100 ml each. The eluate wasanalyzed by the following thin layer chromatographic (TLC) method:adsorbent: Kieselgel 60 F 254 DC (Merck) aluminum foil; developingsolvent: acetone-benzene-acetic acid (50:50:3) mixture; detection: withphosphomolybdic acid reagent. The R_(f) value of pravastatin is 0.5.Fractions containing the product were combined and the acetone wasdistilled off in vacuum. The pH of the 400 ml concentrate was adjustedto 3.5-4.0 by 15% sulfuric acid, then it was extracted three times by150 ml ethyl acetate. The ethyl acetate extracts were combined and driedwith anhydrous sodium sulfate. Subsequently, pravastatin lactone wasprepared from pravastatin acid by adding at room temperature undercontinuous stirring trifluoroacetic acid in catalytic amount. Theformation of pravastatin lactone was controlled by TLC (the R_(f) valueof pravastatin lactone in the above TLC system is 0.7). After thecompletion of the lactone formation, the ethyl acetate was washed with2×50 ml 5% aqueous sodium hydrogen carbonate solution, then washed with50 ml water, dried with anhydrous sodium sulfate and evaporated invacuum. The evaporation residue obtained in a quantity of 3 g wasdissolved in 100 ml acetone and clarified with 0.3 g charcoal. Then thecharcoal was filtered off and the acetone was evaporated in vacuum. Thecrude product obtained was crystallized from 20 ml ethanol. Precipitatedcrystalline pravastatin lactone was filtered off, and washed on thefilter with 30 ml n-hexane and dried at room temperature in vacuum. Inthis way 1.5 g chromatographically pure pravastatin lactone wasobtained. Melting point 140-142° C., [α]_(D)=+194° (c=0.5, methanol).The mother liquor of the crystallization was evaporated in vacuum and1.2 g evaporation residue is obtained, which was chromatographed on 24 gKieselgel 60 adsorbent containing column (diameter of the column: 1.6cm, height of the bed: 20 cm). The crude product dissolved in 5 mlbenzene was layered on the column. For elution mixtures of ethylacetate-n-hexane were used in which the ethyl acetate content wasgradually increased. Pravastatin lactone can be eluted from the columnwith the mixture of 60% ethyl acetate-40% n-hexane. Fractions werecontrolled by TLC using the mixture of ethyl acetate-n-hexane (9:1) asthe developing solvent. The pravastatin lactone-containing fractionswere combined and evaporated in vacuum. According to this method 0.3 gpure product is obtained, its quality identical with that of thepravastatin lactone obtained by crystallization.

The two pravastatin lactone batches were combined and the sodium saltwas prepared according to the following method: 1.8 g pravastatinlactone was dissolved in 20 ml acetone and under stirring 4.5 ml of 1Maqueous sodium hydroxide was added, then the solution was stirred forhalf an hour at room temperature. When the salt formation was completed,20 ml water was added into the mixture and the solution was neutralized,then the acetone was evaporated in vacuum. The aqueous concentrate waschromatographed on a column filled with 150 ml Diaion HP 20 resin(diameter of the column: 2.6 cm, height of the bed: 30 cm). As theeluting agent mixtures of acetone-deionized water were used, where theconcentration of the acetone was increased in 5% steps. Pravastatin canbe eluted from the column by a 15% acetone containing acetone-deionizedwater mixture. Fractions were analyzed by TLC. Fractions containing theproduct are combined and acetone was evaporated in vacuum. Bylyophilization of the aqueous residue 1.3 g pravastatin was obtained.The chromatographically pure product was crystallized from a mixture ofethanol and ethyl acetate.

Melting point: 170-173° C. (decomp.); [α]^(D) ₂₀=+156°, (c=0.5, inwater). Ultraviolet absorption spectrum (20 μg/ml, in methanol):λ_(max)=231, 237, 245 nm; (log ε—4.263; 4.311; 4.136); Infraredabsorption spectrum (KBr): υOH 3415, υCH 2965, υC═O 1730, υCOO⁻ 1575cm⁻¹. ¹H-NMR spectrum (D₂O, δ, ppm): 0.86, d, 3H (2-CH₃); 5.92, dd,J=10.0 and 5.4 Hz, 1H (3-H); 5.99, d, J=10.0 Hz, 1H (4-H); 5.52, br 1H(5H); 4.24, m 1H (6-H); 5.34, br, 1H (8-H); 4.06, m, 1H (β-H), 3.65, m,1H (δ-H); 1.05, d, 3H (2′-CH₃); 0.82, t, 3H (4′-H₃). ¹³C-NMR spectrum(D₂O, δ, ppm): 15.3, q (2-CH₃); 139.5, d (C-3); 129.5, d, (C-4); 138.1,s (C-4a), 127.7, d (C-5); 66.6, d (C-6); 70.1, d (C-8); 182.6 s (COO⁻);72.6. d (C-β); 73.0, d (C-δ); 182.0, s (C-1′) 18.8; q (2′-CH₃); 13.7, q(C-4′). Positive FAB mass spectrum (characteristic ions): [M+Na]⁺ 469;[M+H]⁺ 447. Negative FAB mass spectrum (characteristic ions): [M−H]⁻445, [M−Na]⁻ 423, m/z 101 [2-methyl-butyric acid-H]⁻.

Example 3

In a laboratory scale fermenter with 5 liters working volume,bioconversion culture medium MU/4 was prepared as described in Example1, although it was loaded up to 4.5 liters, the composition of theculture medium was calculated to 5 liters. Then it was sterilized for 45min at 121° C. and inoculated with 500 ml of the inoculum culture madeaccording to Example 1. The fermentation was carried out at 25° C. bythe application of a stirring rate of 300 rpm and an aeration rate of 50liters/hour for 4 days. After 5 g compactin substrate feeding to theculture the bioconversion was carried out according to the Example 2.

After finishing the bioconversion, the 4.9 liters broth, which contained660 μg/ml pravastatin, was filtered and the separated mycelium waswashed by suspension in 2×1 liter deionized water. The pH of thecombined 5.6 liters filtrate of the broth was adjusted by 20% sulfuricacid to 3.5-3.7, then the acidic filtrate was stirred with 2750 ml ethylacetate for 30 min. Subsequently, the phases are separated. The aqueousphase was extracted again with 2×1375 ml ethyl acetate. 470 ml deionizedwater was added to the combined 4740 ml ethyl acetate extract, then thepH of the aqueous ethyl acetate mixture was adjusted to 7.5-8.0 by 1Msodium hydroxide. After 20 min stirring the phases were separated, thenthe ethyl acetate extract was extracted with 2×235 ml deionized water asdescribed above. Then the combined weakly alkaline aqueous solution of1080 ml volume was concentrated in vacuum to 280 ml volume. Theconcentrated aqueous solution was layered on a chromatographic column(ratio of height:diameter=6.5) filled with 280 ml Diaion HP-20(Mitsubishi Co., Japan) non-ionic resin. The adsorption on the columnwas carried out with a flow rate of 250-300 ml/hour, then the column waswashed with 840 ml deionized water. Subsequently, the column was elutedin the following order with 800 ml 5%, 1000 ml 10%, 500 ml 15% and 500ml 20% acetone-containing water. In the course of the elution 50 mlfractions were collected, which were analyzed by the TLC method given inthe Example 2. Fractions containing pravastatin as the main componentwere combined and the obtained solution was concentrated in vacuum to260 ml volume. The concentrated aqueous solution was chromatographed ona column containing Diaion HP-20 resin in 260 ml volume. After theadsorption of pravastatin the column was washed with 790 ml deionizedwater, then eluted with aqueous acetone solutions in 260-260 ml portionsgradually increasing the acetone content as follows: 2.5, 5.0, 7.5,10.0, 12.5, 15.0 and 20.0%. In the course of the column chromatography25 ml fractions were collected, and the pravastatin content of thefractions was analyzed as given before. Fractions containing pravastatinas the single component by TLC were combined and evaporated in vacuum.Subsequently, 0.3 g charcoal was added to the concentrated aqueoussolution (about 30 ml) and pravastatin was clarified at room temperaturefor 30 min. Then the charcoal was removed by filtration from thesolution and the filtrate was lyophilized. In this way 1.62 gpravastatin was obtained in lyophilized form.

Example 4

From the slant culture of the Mortierella maculata nov. spec. E-97[NCAIM(P)F 001266] strain cultivated for 10-12 days, a spore suspensionwas prepared with 5 ml sterile 0.9% sodium chloride solution, and thissuspension was used to inoculate 500 ml VHIG inoculum medium beingsterilized in 3000 ml Erlenmeyer flask.

Composition of the Medium VHIG:

glucose 30 g  meat extract 8 g yeast extract 1 g Tween-80(polyoxyethylene (20) sorbitan monooleate) 0.5 g   in 1000 ml tap water.

Before the sterilization the pH of the medium was adjusted to 7.0 andthe sterilization was carried out at 121° C. for 25 min. The culture wascultivated for 3 days on a rotary shaker (250 rpm, amplitude 2.5 cm),then the obtained inoculum culture was used to inoculate a laboratoryscale fermenter containing bioconversion culture medium PK in 5 litersworking volume.

Composition of the Medium PK:

glucose 40 g  peptone 5 g soybean meal 20 g  K₂HPO₄ 2 g KH₂PO₄ 1 g NaNO₃2 g KCl 0.5 g   in 1000 ml tap water.

Before the sterilization the pH of the medium is adjusted to 7.0. Afterthe inoculation, cultivation, the substrate feeding and bioconversionwere carried out according to Example 2, then the pravastatin wasisolated from the broth in which its concentration was 650 μg/ml at theend of the fermentation.

Finishing the fermentation, the pH of the 4.9 liters broth containing650 μg/ml pravastatin was adjusted under continuous stirring with 2Msodium hydroxide to 9.5-10.0, then after one hour stirring the pH isadjusted to 3.5-3.7 with 20% sulfuric acid. Subsequently, the acidicsolution was extracted with 2.45 liters ethyl acetate. The phases areseparated, and with centrifugation a clear extract was prepared from theemulsified organic phase. The broth was extracted again with 2×1.22liters ethyl acetate by the method given above. The ethyl acetateextracts were combined and 0.4 liters deionized water were added, thenthe pH of the mixture was adjusted to 8.0-8.5 with 1M sodium hydroxide.Phases were separated, and the ethyl acetate phase was extracted with2×0.2 liters deionized water of pH 8.0-8.5 as given above. The pH of thecombined pravastatin containing weakly alkaline aqueous solution wasadjusted under stirring with a 20% sulfuric acid solution to 3.5-3.7.The acidic solution obtained was extracted with 4×0.2 liters ethylacetate. The combined ethyl acetate extracts are washed with 2×0.2liters deionized water, then 150 mole % dibenzylamine—calculated for thepravastatin content measured by HPLC—was added into the ethyl acetatesolution. The ethyl acetate solution was concentrated in vacuum to 0.2liters volume. Further 20 mole % dibenzylamine was added to theconcentrate obtained, and the precipitated solution was kept overnightat 0-5° C. The precipitated pravastatin dibenzylamine salt was filtered,then the precipitate was washed on the filter with cold ethyl acetateand then two times with n-hexane, and finally it is dried in vacuum at40-50° C. The crude product obtained (3.9 g) was dissolved in 100 mlmethanol at room temperature, then the solution was clarified by 0.45 gcharcoal. Thereafter the methyl alcoholic filtrate is concentrated invacuum. The evaporated residue was dissolved in 120 ml acetone at anexternal temperature of 62-66° C., then the solution was cooled to roomtemperature. Subsequently, the recrystallization was continued overnightat 0-5° C. Precipitated crystals were filtered, then the crystals werewashed on the filter two times with cold acetone and two times withn-hexane. The recrystallized pravastatin dibenzylamine salt wassuspended in the mixture of 160 ml isobutyl acetate and 80 ml deionizedwater. Subsequently, sodium hydroxide was added in an equivalent amountinto the suspension under stirring. After the disappearance of thesuspension the phases were separated and the pravastatin containingaqueous solution was washed with 2×30 ml isobutyl acetate. The aqueoussolution obtained was clarified with charcoal. Then the aqueous filtratewas concentrated to about 20 ml volume. The aqueous solution obtainedwas loaded on a chromatographic column (height:diameter=22) filled with0.4 liters Sephadex LH-20 gel (supplier: Pharmacia, Sweden). In thecourse of the chromatography deionized water was used as the eluent, and20 ml fractions were collected. Fractions were analyzed by TLC, thenthose containing pravastatin also by HPLC using the methods describedabove. Fractions containing pure pravastatin were combined andlyophilized. In this way 1.75 g pravastatin was obtained, the purity ofwhich is higher than 99.5% by HPLC.

Example 5

A spore suspension was prepare& from the slant culture of theMortierella maculata n. spec. E-97 [NCAIM(P)F 001266] strain cultivatedfor 10-12 days with 5 ml sterile 0.9% sodium chloride solution, and then500 ml inoculum medium was inoculated with it as described in Example 4.In a laboratory scale fermenter with 5 liters working volumebioconversion culture medium PC/4 is sterilized for 45 min at 121° C.and then inoculated with the seed culture.

Composition of the Medium PC/4:

malt extract 5.0% soybean meal 1.0% peptone 1.0% corn steep liquor 1.0%MgSO₄ × 7 H₂O 0.1% in 1000 ml tap water.

Before the sterilization the pH of the medium is adjusted to 7.0. Afterthe inoculation, the cultivation and substrate feeding were carried outaccording to the Example 2, and then 5.1 liters broth with aconcentration of 610 μg/ml pravastatin was obtained.

From the broth 3.7 g pravastatin dibenzylamine salt crude product wasproduced by the method given in Example 4, from which afterrecrystallization 2.9 g pravastatin dibenzylamine salt was obtained. Therecrystallized pravastatin dibenzylamine salt was suspended in 45 mlethanol, then under stirring 110 mole % sodium hydroxide was added bythe feeding of 1M ethanolic sodium hydroxide solution. Stirring of thesolution is continued for half an hour, then 0.3 g charcoal was addedinto it and stirred for another half an hour. The solution was filtered,and the filtrate was concentrated to 15 ml. Then 60 ml acetone was addedto the concentrate at 56-60° C. The solution obtained was cooled to roomtemperature, then kept overnight at +5° C. Subsequently, the precipitatewas filtered, then washed with 2×20 ml acetone, 2×20 ml ethyl acetateand 2×20 ml n-hexane, and finally dried in vacuum. The resulting 1.7 gcrude pravastatin was dissolved in ethanol, then clarified with charcoaland crystallized from an ethanol-ethyl acetate mixture. In this way 1.54g pravastatin was obtained that was identical with the product ofExample 2.

Example 6

As described in Example 4, from the slant culture of the Mortierellamaculata n. spec. E-97 [NCAIM(P)F 001266] strain cultivated for 7-10days, a 500 ml inoculum medium MI sterilized in a 3000 ml Erlenmeyerflask was inoculated and incubated at 28° C. for 3 days on a rotaryshaker.

Composition of the Medium MI:

glucose  40 g casein   5 g KCl 0.5 g NaNO3   3 g KH2PO4   2 g MgSO₄ ×7H₂O 0.5 g FeSO₄ × 7H₂O 0.01 g  in 1000 ml tap water.

Before the sterilization the pH of the medium is adjusted to 6.0 and thesterilization is carried out at 121° C. for 35 min. The seed cultureobtained is inoculated into 5 liters bioconversion medium P12 sterilizedin a fermenter.

Composition of the Medium P12:

glucose 10 g malt extract 50 g yeast extract  5 g corn steep liquor  5 gMgSO₄ × 7H₂O  1 g Tween-80 0.5 g  in 1000 ml tap water.

Before the sterilization the pH of the medium is adjusted to 7.0, thenthe sterilization was carried out at 121° C. for 45 min. Thefermentation, substrate feeding and bioconversion were carried outaccording to the Example 2. After finishing the bioconversion thepravastatin formed in the concentration of 620 μg/ml was isolated asfollows:

The pH of 5.15 liters broth containing 620 μg/ml pravastatin wasadjusted with 2M sodium hydroxide to 9.5 value then stirred at roomtemperature for 1 hour. The broth was filtered and the mycelium waswashed with suspension in 1×2 liters and then 1×0.5 liters water.Filtrates are combined and the pH of the aqueous solution was adjustedwith 20% sulfuric acid to 3.7 value and extracted with 2.5 liters thenwith 1.5 liters ethyl acetate. The ethyl acetate extracts were combined,washed with 2×0.5 liters water and 1.95 g dicyclohexylamine was added.The ethyl acetate extract was concentrated at 40° C. to 200 ml underreduced pressure, and 0.195 g dicyclohexylamine was added again into theconcentrate, which was then stirred at 15° C. for 6 hours. Theprecipitated crystalline material was filtered, washed with 20 ml andwith 15 ml ethyl acetate and dried at 40° C. In this way 3.51 g crudeproduct was obtained. After the recrystallization of the crude productin an acetone-ethanol mixture, 3.05 g of pravastatin dicyclohexylaminesalt was obtained (melting point: 162-168° C.), which was converted topravastatin according to the Example 5.

Example 7

The fermentation, substrate feeding and bioconversion were carried outwith the Mortierella maculata n. spec. E-97 [NCAIM(P)F 001266] strain asdescribed in Example 2. Pravastatin obtained as a result of thebioconversion is isolated from the broth as follows.

5 liters culture broth containing in concentration 650 μg/ml pravastatinwas filtered on a filter cloth. The mycelium of the mold was stirred in2 liters 0.1M sodium hydroxide solution for an hour, then filtered. Thetwo filtrates were combined and the pH was adjusted with 15% sulfuricacid to 3.5-4.0. Subsequently, the solution was extracted with 2×1.8liters ethyl acetate. The combined ethyl acetate phases were washed with800 ml water. Then 400 ml deionized water was added and the pH of themixture is adjusted by 1M sodium hydroxide to a 8.0-8.5 value. Themixture was stirred for 15 minutes, then the phases were separated. 300ml water was added to the ethyl acetate phase and the pH are adjusted to8.0-8.5. After stirring for 15 minutes the phases were separated. 300 mlwater was added again to the ethyl acetate phase and the pH was adjustedto 8.0-9.5. Then the mixture was stirred for 15 min. The two phases wereseparated again. All aqueous phases were combined and the pH areadjusted with 15% sulfuric acid to 3.5-4.0, then extracted with 3×300 mlethyl acetate. The combined ethyl acetate extracts were washed with 150ml water, dried with anhydrous sodium sulfate, and filtered. Then 150mole % dioctylamine—calculated for the pravastatin content—was added tothe ethyl acetate extract. The ethyl acetate was evaporated to about{fraction (1/10)} volume and acetone was added until precipitation. Themixture was kept at +5° C. overnight. The precipitate was filtered on aG-4 filter, washed with 20 ml acetone and then with 20 ml n-hexane anddried in vacuum at room temperature. The 3.3 g crude pravastatindioctylamine salt obtained was recrystallized from 20 ml acetoneresulting in 2.7 g pure pravastatin dioctylamine salt. Melting point:143-146° C. The pravastatin dioctylamine salt was converted topravastatin with the method given in Example 5.

Example 8

By the development of the hydroxylation ability of Mortierella maculatan. spec. E-97 strain isolated from natural habitat, which is able to6β-hydroxylate compactin, in the mutation-selection and enzyme inductionexperiments discussed in detail below, Mortierella maculata n. sp.E-97/15/13 [NCAIM(P)F 001267] mutant strain was produced.

Mortierella maculata n. sp. E-97 [NCAIM(P)F 001266] strain isolated byus was cultivated on MS slant agar medium at 28° C. for 7 days.

Composition of Agar Medium MS:

glucose  4 g malt extract 10 g yeast extract  4 g agar 20 g in 1000 mldistilled water.

Spores were washed off from the slant cultures by 5 ml 0.9% sodiumchloride solution, then after transferring the spore suspension into asterile Petri dish it was irradiated by ultraviolet light for 1 minute.Subsequently, N-methyl-N′-nitro-N-nitrosoguanidine was added to thespore suspension in the final concentration of 2000 μg/ml. Then thesuspension was transferred into a 100 ml Erlenmeyer flask and it wasshaken at 28° C. with a rate of 150 rpm for 20 min. Subsequently, thespores were sedimented by centrifugation with a rate of 4000 rpm for 10min, then suspended in sterile 0.9% sodium chloride solution. Thesuspension was spread on an agar plate MU-VB containing 10 μg/ml benomyland 1% defibrillated blood.

Composition of Agar Medium MU-VB:

glucose 40 g asparagine  2 g peptone 2.5 g  potassium dihydrogenphosphate 0.5 g  agar 20 g

in 990 ml distilled water; after sterilization the medium was completedwith 10 ml bovine blood and 10 mg benomyl.

The agar plates were incubated at 28° C. for 7 days, then the growncolonies were transferred by random selection into test tubes containingagar medium PS.

Composition of Agar Medium PS:

glucose 40 g mycological peptone 10 g agar 15 g in 1000 ml distilledwater.

Before sterilization the pH of the medium is adjusted to 5.6-5.7 value.The sterilization is carried out at 121° C. for 20 min.

Slant cultures were incubated at 28° C. for 12 days, and theirpravastatin productivity was tested in shaken flask experiments asdescribed in Example 1. Mortierella maculata n. sp. E-97/15/13 mutantstrain was selected by this method, which yielded pravastatin exceeding60% conversion rate from the applied compactin acid sodium saltsubstrate being in the concentration of 1000 μg/ml.

The hydroxylase enzyme of Mortierella maculata n. sp. E-97/15/13 strainwas induced by the cultivation on MU-VB agar medium containing 100 μg/ml8-de-(2-methyl-butyryl) compactin and/or compactin. After randomselection of the grown colonies they were transferred into inducercontaining MU-VB slants. Pravastatin productivity of the grown slantcultures was examined by the method written in the Example 1 with thedifference that the compactin substrate feeding in the quantity of 500μg/ml was carried out from the 4th day of the fermentation for further11 days and the compactin sodium substrate was added gradually duringthe twelve days converted completely to pravastatin. By the end of thebioconversion carried out in 50 shake flask cultures from 30 g compactinsodium substrate the formation of 18.5 g pravastatin was measured byHPLC. Recovery of the pravastatin from the combined fermentation brothswas carried out according to the following method.

After finishing the fermentation the pH of 5.5 liters broth with apravastatin concentration of 3360 μg/ml was adjusted with 20% sulfuricacid solution to 3.5-3.7. Subsequently, the acidic solution wasextracted by 2.75 liters ethyl acetate. Phases were separated, and aclear extract was prepared by centrifugation from the emulsified organicphase. Broth was extracted two more times with 1.37 liters ethyl acetateas previously described. The combined ethyl acetate extracts were washedwith 2×1.15 liters deionized water, then 150 mole %dibenzylamine—calculated for the pravastatin content measured byHPLC—was added to the ethyl acetate solution. The ethyl acetate solutionwas concentrated in vacuum to about 0.23 liters volume. Further 20 mole% dibenzylamine was added to the concentrate and the precipitatesolution was kept overnight at 0-5° C. Precipitated pravastatin aciddibenzylamine salt was filtered, then the precipitate was washed bysuspending it in cooled ethyl acetate and then two times in n-hexane,finally dried at 40-50° C. in vacuum. The crude product obtained (22.4g) was dissolved in 0.67 liters acetone at 62-66° C. temperature, andthe solution was clarified with 2.2 g charcoal. After the clarificationthe acetone filtrate was concentrated in vacuum to 0.56 liters volume.Crystals precipitated from the concentrate were dissolved again at theabove temperature, then the solution was cooled to room temperature.Subsequently, the recrystallization was continued overnight at 0-5° C.Precipitated crystals were filtered, and washed by suspension two timesin cooled acetone and two times in n-hexane. Recrystallized pravastatinacid dibenzylamine salt was dried in vacuum at 40-50° C. Pravastatinacid dibenzylamine salt obtained (14.8 g) was dissolved at 40-44° C. in740 ml ethyl acetate-ethanol (9:1) mixture, then 110 mole % sodiumhydroxide was added to the solution in form of a 1M ethanolic solution.Stirring of the obtained precipitated solution was continued for half anhour at room temperature, then a complete precipitation was achieved asa result of the application or ice cooling for 1-1.5 hours.Subsequently, the precipitate was filtered and washed with 2×150 mlcooled ethyl acetate and 2×150 ml n-hexane, finally dried in vacuum at40-50° C. The pravastatin obtained was dissolved in ethanol, clarifiedby 1.0 g charcoal, then crystallized from ethanol-ethyl acetate mixture.This way 9.4 g pravastatin was obtained, with physical constantscorresponding to the data given in Example 2.

Although certain presently preferred embodiments of the invention havebeen described herein, it will be apparent to those skilled in the artto which the invention pertains that variations and modifications of thedescribed embodiments may be made without departing from the spirit andscope of the invention. Accordingly, it is intended that the inventionbe limited only to the extent required by the appended claims and theapplicable rules of law.

We claim:
 1. A microbial process for the preparation of a compound offormula (I)

from a substrate of formula (II)

wherein R stands for an alkali metal or ammonium ion, comprising thesteps of a) cultivating a strain of Mortierella maculata filamentousmold species able to 6β-hydroxylate a compound of formula (II) on or ina nutrient medium containing assimilable carbon- and nitrogen sourcesand mineral salts, b) feeding the substrate to be transformed into theculture of Mortierella maculata, c) fermenting the substrate until theend of bioconversion, and d) isolating the compound of formula (I) fromthe culture.
 2. The process of claim 1 wherein the strain of Mortierellamaculata is cultivated at about 25° C. to 30° C.
 3. The process of claim2 wherein the hydroxylase activity of the Mortierella maculata isinduced by exposure to 8-de-(2-methylbutyryl)-compactin or compactinduring cultivation.
 4. The process of claim 1 wherein the strain ofMortierella maculata is the Mortierella maculata n. sp. E-97 straindeposited at the National Collection of Agricultural and IndustrialMicroorganisms, Budapest, Hungary under the number NCAIM(P)F
 001266. 5.The process of claim 1 wherein the strain of Mortierella maculata is theMortierella maculata n. sp. E-97/15/13 strain deposited at the NationalCollection of Agricultural and Industrial Microorganisms, Budapest,Hungary under the number NCAIM(P)F
 001267. 6. The process of claim 1wherein a source of assimilable carbon selected from the groupconsisting of glucose, fructose and glycerine is added to the culture.7. The process of claim 1 wherein a source of assimilable nitrogenselected from the group consisting of soybean meal, peptone, casein,yeast extract and meat extract is added to the culture.
 8. The processof claim 1 wherein the nutrient medium is a liquid culture broth.
 9. Theprocess of claim 8 wherein the substrate is fed into the culture brothwhen the pH of the culture broth is equal to or greater than 6.3 andwherein the substrate is fed into the culture broth contemporaneouslywith addition of a source of assimilable carbon and wherein the amountof the source of assimilable carbon that is added is about 1% by weightper volume of the culture broth if the pH is higher than 6.7, andwherein the amount of the source of assimilable carbon that is added isabout 0.5% by weight per volume of the culture broth if the pH is withinthe range of 6.3 to 6.7.
 10. The process of claim 8 wherein theisolating comprises separating pravastatin free acid from the liquidculture broth by adsorption on an anionic ion exchange resin.
 11. Theprocess of claim 8 wherein the isolating comprises: a) extractingpravastatin free acid from the culture broth into a water immiscibleorganic solvent, b) either lactonizing the pravastatin free acid orconverting the pravastatin free acid to a secondary amine salt, and c)converting the lactone or secondary amine salt to the compound offormula (I).
 12. The process of claim 11 wherein the culture broth isadjusted to pH 3.5-3.7 before extracting the culture broth with thewater-immiscible organic solvent.
 13. The process of claim 12 whereinthe water-immiscible organic solvent is ethyl acetate.
 14. The processof claim 12 wherein the water immiscible organic solvent is isobutylacetate.
 15. The process of claim 8 wherein isolating the compound offormula (I) comprises: a) extracting the culture broth with awater-immiscible organic solvent, b) contacting the organic extract withan alkaline aqueous solvent containing sodium to form the compound offormula (I) as a solute in the aqueous solvent, and c) purifying thecompound of formula (I) by chromatography on a non-ionic adsorbingresin.
 16. The process of claim 15 wherein the alkaline aqueous solventcontaining sodium is aqueous sodium hydroxide.
 17. The process of claim8 wherein isolating the compound of formula (I) comprises: a) filteringthe culture broth, b) loading the filtered culture broth onto an anionicexchange resin, c) eluting pravastatin free acid from the resin, d)lactonizing the pravastatin free acid, e) isolating pravastatin lactone,f) hydrolyzing the lactone with sodium hydroxide to form the compound offormula (I), and g) purifying the compound of formula (I) bychromatography on a non-ionic adsorption resin.
 18. The process of claim17 wherein the anion exchange resin has quaternary ammonium activegroups carried on a polystyrene-divinylbenzene skeleton.
 19. The processof claim 8 wherein the substrate is fed into the culture broth when thepH of the culture broth is equal to or greater than 6.3, and wherein asufficient amount of assimilable carbon is added to the culture broth tomaintain the pH at about 6.3 to about 6.7.
 20. The process of claim 19wherein the assimilable carbon consists essentially of glucose.
 21. Theprocess of claim 12 wherein the pravastatin free acid is converted to asecondary amine salt and the converting of the pravastatin free acid toa secondary amine salt comprises: a) adding a secondary amine containingalkyl-, cycloalkyl-, aralkyl- or aryl substituents to thewater-immiscible organic solvent after the extracting, and b)precipitating the secondary amine salt of pravastatin.
 22. The processof claim 21 wherein converting the secondary amine salt to the compoundof formula (I) comprises: a) suspending the precipitated secondary aminesalt of pravastatin in a mixture of water and an ester having theformula CH₃COOR′, where R′ is an alkyl group containing 1-4 carbonatoms, b) contacting an aqueous solution of sodium hydroxide with thesuspension, c) washing the aqueous solution with isobutyl acetate, d)clarifying the aqueous solution with activated carbon, and e)lyophilizing the solution to obtain the compound of formula (I).
 23. Theprocess of claim 22 wherein the 1-4 carbon atom-containing alkyl esteris isobutyl acetate.
 24. The process of claim 22 wherein converting thesecondary amine salt to the compound of formula (I) further comprisespurifying the compound of formula (I) to at least 99.5%, as measured byHPLC, using gel chromatography.
 25. The process of claim 21 whereinconverting the secondary amine salt to the compound of formula (I)further comprises: a) suspending the precipitated secondary amine saltof pravastatin in a 1-4 carbon atom-containing alcohol, b) adding asufficient amount of ethanolic solution of sodium hydroxide to thesuspension to dissolve the secondary amine salt of pravastatin, c)adding acetone to the resulting solution to precipitate the compound offormula (I), and d) separating the compound of formula (I).
 26. Theprocess of claim 25 wherein the 1-4 carbon atom-containing alcohol isethanol.
 27. The process of claim 21 wherein converting the secondaryamine salt to the compound of formula (I) further comprises: a)dissolving the precipitated secondary amine salt of pravastatin in amixture of a 1-4 carbon atom-containing alkyl ester of a 1-4 carbonatom-containing alkane carboxylic acid and a 1-4 carbon atom-containingalcohol, and b) adding sodium hydroxide to the resulting solution toprecipitate the compound of formula (I), and c) separating the compoundof formula (I).
 28. The process of claim 27 wherein the mixture is amixture of ethyl acetate and ethanol.
 29. The process of claim 21wherein the secondary amine is selected from the group consisting ofdibenzyl amine, dicyclohexyl amine and dioctyl amine.